Vaccines and immunotherapeutics using codon optimized IL-15 and methods for using the same

ABSTRACT

Nucleic acid molecules that encode IL-15 or fragments thereof, which express protein at a higher level than nucleic acid molecules with native coding sequences for IL-15 are disclosed. Nucleic acid molecules with additional modifications such as the absence of coding sequences for IL-15 signal sequences and/or the absence of IL-15 untranslated sequences and/or inclusion of non-IL-15 signal sequences are also disclosed. Vectors, including plasmids and viral vectors, comprising such nucleic acid molecules; and to host cells comprising such nucleic acid molecules are disclosed as well as methods of using such nucleic acid molecules alone or in combination with nucleic acid sequences encoding immunogens which are part of the nucleic acid molecules and/or part of a different nucleic acid molecule. Recombinant vaccines and live attenuated pathogens encoding fusion proteins, and methods of using the same, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage filing of InternationalApplication Serial No. PCT/US2007/000886 filed Jan. 12, 2007, whichclaims priority to U.S. Provisional Patent Application No. 60/758,856,filed Jan. 13, 2006, each of which is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules that include acodon optimized nucleic acid sequence that encodes IL-15 and fragmentsthereof, improved vaccines, improved methods for prophylactically and/ortherapeutically immunizing individuals against immunogens, and toimproved immunotherapeutic compositions and improved immunotherapymethods.

BACKGROUND OF THE INVENTION

Immunotherapy refers to modulating a person's immune responses to imparta desirable therapeutic effect. Immunotherapeutics refer to thosecompositions which, when administered to an individual, modulate theindividual's immune system sufficient to ultimately decrease symptomswhich are associated with undesirable immune responses or to ultimatelyalleviate symptoms by increasing desirable immune responses. In somecases, immunotherapy is part of a vaccination protocol in which theindividual is administered a vaccine that exposes the individual to animmunogen against which the individual generates an immune response. insuch cases, the immunotherapeutic increases the immune response and/orselectively enhances a portion of the immune response (such as thecellular arm or the humoral arm) which is desirable to treat or preventthe particular condition, infection or disease.

Vaccines are useful to immunize individuals against target antigens suchas allergens, pathogen antigens or antigens associated with cellsinvolved in human diseases. Antigens associated with cells involved inhuman diseases include cancer-associated tumor antigens and antigensassociated with cells involved in autoimmune diseases.

In designing such vaccines, it has been recognized that vaccines thatproduce the target antigen in cells of the vaccinated individual areeffective in inducing the cellular arm of the immune system.Specifically, live attenuated vaccines, recombinant vaccines which useavirulent vectors, and DNA vaccines each lead to the production ofantigens in the cell of the vaccinated individual which results ininduction of the cellular arm of the immune system On the other hand,killed or inactivated vaccines, and sub-unit vaccines which compriseonly proteins do not induce good cellular immune responses although theydo induce a humoral response.

A cellular immune response is often necessary to provide protectionagainst pathogen infection and to provide effective immune-mediatedtherapy for treatment of pathogen infection, cancer or autoimmunediseases. Accordingly, vaccines that produce the target antigen in cellsof the vaccinated individual such as live attenuated vaccines,recombinant vaccines that use avirulent vectors and DNA vaccines areoften preferred.

While such vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines. There is a need forcompositions and methods that produce an enhanced immune response.

Likewise, while some immunotherapeutics are useful to modulate immuneresponse in a patient there remains a need for improvedimmunotherapeutic compositions and methods.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid molecules that comprisesnucleic acid sequence that encodes IL-15 protein comprising SEQ ID NO:1or a fragment thereof which encodes a functional fragment of IL-15.

The present invention relates to nucleic acid molecules comprising SEQID NO:1 or a fragment thereof which encodes a functional fragment ofIL-15 that are free of coding sequence for an IL-15 signal sequenceand/or free of IL-15 Kozak region and/or IL-15 5′ untranslated regionand/or IL-15 3′ untranslated region and/or comprising a coding sequencefor a non-IL-15 signal sequence.

The present invention relates to nucleic acid molecules that comprisesnucleic acid sequence that encodes IL-15 protein comprising SEQ ID NO: 1or a fragment thereof which encodes a functional fragment of IL-15 andfurther comprise coding sequence for an immunogen.

The present invention relates to compositions that comprise a nucleicacid molecule that comprises nucleic acid sequence comprising SEQ IDNO:1 or a fragment thereof which encodes a functional fragment of IL-15and a nucleic acid molecule that comprises a nucleic acid sequence thatencodes an immunogen.

The present invention further relates to methods of modulating an immuneresponse in an individual comprising administering to said individual anucleic acid molecule comprising SEQ ID NO:1 or a fragment thereof whichencodes a functional fragment of IL-15.

The present invention further relates to recombinant vaccines thatcomprise a nucleic acid molecule comprising SEQ ID NO: 1 or a fragmentthereof which encodes a functional fragment of IL-15.

The present invention relates to methods of inducing an immune responsein an individual against an immunogen comprising administering to saidindividual a nucleic acid molecule comprising SEQ ID NO:1 or a fragmentthereof which encodes a functional fragment of IL-15 as part or incombination a nucleic acid molecule that encodes an immunogen or incombination with an immunogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEQ ID NO: 1.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

As used herein the term “target protein” is meant to refer to peptidesand protein encoded by gene constructs of the present invention that actas target proteins for an immune response. The terms “target protein”and “immunogen” are used interchangeably and refer to a protein againstwhich an immune response can be elicited. The target protein is animmunogenic protein that shares at least an epitope with a protein fromthe pathogen or undesirable cell-type such as a cancer cell or a cellinvolved in autoimmune disease against which an immune response isdesired. The immune response directed against the target protein willprotect the individual against and/or treat the individual for thespecific infection or disease with which the target protein isassociated.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence that encodes a targetprotein or immunomodulating protein. The coding sequence includesinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of the individual to whom the nucleicacid molecule is administered.

As used herein, the term “expressible form” refers to gene constructsthat contain the necessary regulatory elements operable linked to acoding sequence that encodes a target protein or an immunomodulatingprotein, such that when present in the cell of the individual, thecoding sequence will be expressed.

As used herein, the term “sharing an epitope” refers to proteins thatcomprise at least one epitope that is identical to or substantiallysimilar to an epitope of another protein.

As used herein, the term “substantially similar epitope” is meant torefer to an epitope that has a structure that is not identical to anepitope of a protein but nonetheless invokes a cellular or humoralimmune response which cross reacts to that protein.

As used herein, the term “intracellular pathogen” is meant to refer to avirus or pathogenic organism that, at least part of its reproductive orlife cycle, exists within a host cell and therein produces or causes tobe produced, pathogen proteins.

As used herein, the term “hyperproliferative diseases” is meant to referto those diseases and disorders characterized by hyperproliferation ofcells.

As used herein, the term “hyperproliferative-associated protein” ismeant to refer to proteins that are associated with a hyperproliferativedisease.

As used herein the term “immunomodulating protein” refers to a proteinthat modulates the immune system of a person to whom theimmunomodulating protein is delivered. Examples of immunomodulatoryproteins include: IL-15, CD40L, TRAIL; TRAILrecDRC5, TRAIL-R2, TRAIL-R3,TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, F461811 or MICA,MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, CD30, CD153 (CD30L), Fos,c-jun, Sp-1, Ap1, Ap-2, p38, p65Rel,MyD88, IRAK, TRAF6, IkB, NIK, SAP K,SAP1, JNK2, JNK1B2, JNK1B1, JNK2B2, JNK2B1, JNK1A2, JNK2A1, JNK3A1,JNK3A2, NF-kappa-B2, p49 splice form, NF-kappa-B2, p100 splice form,NF-kappa-B2, p105 splice form, NF-kappa-B 50K chain precursor, NFkB p50,human IL-1 α, human IL-2, human IL-4, murine IL-4, human IL-5, humanIL-10, human IL-15, human IL-18, human TNF-α, human TNF-β, humaninterleukin 12, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL4, CSF,G-CSF, GM-CSF, M-CSF, LFA-3, ICAM-3, ICAM-2, ICAM-1, PECAM, P150.95,Mac-1, LFA-1, CD34, RANTES, IL-8, MIP-1α, E-selecton, CD2, MCP-1,L-selecton, P-selecton, FLT, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP,Apo-3, AIR, LARD, NGRF, DR4(TRAIL), DR5, KILLER, TRAIL-R2, TRICK2, DR6,ICE, VLA-1, and CD86 (B7.2).

Overview

The invention provides a nucleic acid sequence that encodes IL-15 whichprovides improved protein expression relative to the native sequence.The improved IL-15 coding sequence can be used in combination with thediscoveries set forth in PCT application PCT/US04/18962 filed Jun. 14,2004, U.S. Provisional Application No. 60/478,210 filed Jun. 13, 2003and U.S. Provisional Application No. 60/478,205 filed Jun. 13, 2003,which are incorporated herein by reference, particularly those providingIL-15 protein coding sequences linked to non-IL-15 signal peptide,particularly IgE signal peptide, and the use of such constructs invaccines and in constructs for delivery of IL-15 protein as animmunomodulating protein. In some preferred embodiments, the inventionprovides vectors, vaccines and immunomodulatory compositions and methodscomprising nucleic acid molecules that comprise the nucleotide sequenceof SEQ ID NO:1 or fragments thereof that encode functional fragments ofIL-15. In some preferred embodiments, such nucleic acid molecules areprovided free of coding sequences for IL-15 signal sequence, ands morepreferably free of the IL-15 Kozak region and untranslated regions. Insome preferred embodiments, the invention provides vectors, vaccines andimmunomodulatory compositions and methods comprising nucleic acidmolecules that comprise SEQ ID NO:1 or fragments thereof that encodefunctional fragments of IL-15 which are linked to coding sequences forhuman IgE signal sequence.

Native sequences that encode IL-15 have been modified to improveexpression. In earlier improvements, elements such as coding sequencesfor IL-15 sequence and untranslated regions were deleted to improveexpression. These earlier improvements may be incorporated and used inconjunction with the improved coding sequence of mature IL-15 proteinset forth in SEQ ID NO:1. In preferred embodiments, the nucleic acidmolecule that includes SEQ ID NO:1 is free of the coding sequence forIL-15 signal peptide, and preferably another signal protein such as IgEsignal protein is provided in its place. Moreover, the IL-15 Kozakregion and untranslated regions are removed as well to eliminateinhibitory elements. The only 11-15 sequences that constructs preferablyinclude are the IL-15 sequences that encode the amino acid sequence ofthe mature IL-15 protein free of IL-15 signal peptide.

According to some embodiments, compositions are provided which comprisean isolated nucleic acid molecule comprising a nucleic acid sequencethat encodes a fusion protein comprising a non-IL-15 signal sequencelinked to IL-15 protein encoded by SEQ ID NO:1 or a fragment thereofthat encodes a functional fragment of IL-15. In some preferredembodiments, the molecule is free of coding sequence for IL-15 signalsequence. In some preferred embodiments, the fusion protein isnon-immunogenic in a human.

According to some embodiments, compositions are provided that include anucleic acid construct comprising SEQ ID NO:1 or a fragment thereof thatencodes a functional fragment of IL-15 and optionally the other earlierimprovements described above may also include on the same nucleic acidmolecule or a different nucleic acid molecule, a nucleic acid sequencethat encodes an immunogen. Generally, immunogens, which are discussedbelow, may be any immunogenic protein including allergens, pathogenantigens, cancer-associated antigens or antigens linked to cellsassociated with autoimmune diseases. In preferred embodiments, theimmunogen is a pathogen antigen, most preferably a pathogen selectedfrom the group consisting of HIV, HSV, HCV, and WNV.

In some preferred embodiments, nucleic acid constructs are plasmids. Insome preferred embodiments, the nucleic acid molecule is incorporated ina viral vector such as vaccinia, adenovirus, adenovirus associatedvirus, retrovirus, RSV, VSV, poxvirus or any other acceptable viralvector useful as a vaccine or gene therapy vector.

Genetic constructs comprising SEQ ID NO:1 or a fragment thereof thatencodes a functional fragment of IL-15 may be incorporated directly intolive attenuated pathogens according to some aspect of the invention.Examples of such pathogens useful as vaccines are set out below. Inpreferred embodiments, human IL-15, preferably free of IL-15 signalsequence, is linked to human IgE signal sequence.

Compositions that include coding sequences for immunogens are useful asvaccines. Compositions that do not include coding sequences forimmunogens may be useful as immunomodulatory compositions. In someembodiments, protein immunogens, are also provided as a target for theimmune response which will be enhanced by the expression of IL-15.

In some preferred embodiments, nucleic acid constructs are plasmids. Insome preferred embodiments, the nucleic acid molecule is incorporated ina viral vector such as vaccinia, adenovirus, adenovirus associatedvirus, retrovirus, or any other acceptable viral vector useful as avaccine or gene therapy vector.

Genetic constructs comprising SEQ ID NO:1 or a fragment thereof thatencodes a functional fragment of IL-15 may be incorporated directly intolive attenuated pathogens according to some aspects of the invention.Examples of such pathogens useful as vaccines are set out below. Inpreferred embodiments, human IL-15, preferably free of IL-15 signalsequence, is linked to human IgE signal sequence

According to some embodiments of the invention, compositions of theinvention comprise genetic constructs including coding sequences forimmunogens and/or immunogenic proteins. Such compositions are deliveredto an individual to modulate the activity of the individual's immunesystem and thereby enhance the immune response against the immunogen.When the nucleic acid molecules that encode an immunomodulatory proteinare taken up by cells of the individual the nucleotide sequences thatencode the immunomodulatory protein are expressed in the cells and theproteins are thereby delivered to the individual. Aspects of theinvention provide methods of delivering the coding sequences of theproteins on a single nucleic acid molecule, in compositions comprisingdifferent nucleic acid molecules that encodes one or more of the varioustranscription factor or intermediate factors, as part of recombinantvaccines and as part of attenuated vaccines.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual against a pathogen or abnormal, disease-relatedcells. The vaccine may be any type of vaccine such as, a live attenuatedvaccine, a cell vaccine, a recombinant vaccine or a nucleic, acid or DNAvaccine.

The present invention relates to compositions for delivering theimmunomodulating proteins and methods of using the same.

The nucleic acid molecules may be delivered using any of several wellknown technologies including DNA injection (also referred to as DNAvaccination), recombinant vectors such as recombinant adenovirus;recombinant adenovirus associated virus and recombinant vaccinia.

DNA vaccines are described in U.S. Pat. Nos. 5,593,972, 5,739,118,5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055,5,676,594, and the priority applications cited therein, which are eachincorporated herein by reference. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are bothincorporated herein by reference.

Routes of administration include, but are not limited to, intramuscular,intransally, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Preferred routes of administration include to mucosal tissue,intramuscular, intraperitoneal, intradermal and subcutaneous injection.Genetic constructs may be administered by means including, but notlimited to, traditional syringes, needleless injection devices, or“microprojectile bombardment gene guns”.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a. functioning extracbromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material in the form of a plasmid orplasmids. Alternatively, linear DNA that can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents that promote DNA integration into chromosomes may beadded. DNA sequences that are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal. Genetic constructsinclude regulatory elements necessary for gene expression of a nucleicacid molecule. The elements include: a promoter, an initiation codon, astop codon, and a polyadenylation signal. In addition, enhancers areoften required for gene expression of the sequence that encodes thetarget protein or the immunomodulating protein. It is necessary thatthese elements be operable linked to the sequence that encodes thedesired proteins and that the regulatory elements are operably in theindividual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation-signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV)such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalthat is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode a target protein, the immunomodulating proteinand, additionally, genes for proteins which further enhance the immuneresponse against such target proteins. Examples of such genes are thosewhich encode other cytokines and lymphokines such as alpha-interferon,gamma-interferon, platelet derived growth factor (PDGF), TNF, GM-CSF,epidermal growth factor (EGF), IL-1, IL-2, 11-4, IL-6, IL-10, IL-12 andIL-15 including IL-15 having the signal sequence deleted and optionallyincluding the signal sequence from IgE.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the geneticconstruct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs that are functional in the cells.

In some embodiments, gene constructs may be provided to in order toproduce coding sequences for the immunomodulatory proteins describedherein linked to IgE signal peptide.

One method of the present invention comprises the steps of administeringnucleic acid molecules intramuscularly, intranasally, intraperatoneally,subcutaneously, intradermally, or topically or by lavage to mucosaltissue selected from the group consisting of inhalation, vaginal,rectal, urethral, buccal and sublingual.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a polynucleotide function enhanceror a genetic vaccine facilitator agent. Polynucleotide functionenhancers are described in U.S. Ser. Nos. 5,593,972, 5,962,428 andInternational Application Serial Number PCT/US94/00899 filed Jan. 26,1994, which are each incorporated herein by reference. Genetic vaccinefacilitator agents are described in U.S. Ser. No. 021,579 filed Apr. 1,1994, which is incorporated herein by reference. The co-agents that areadministered in conjunction with nucleic acid molecules may beadministered as a mixture with the nucleic acid molecule or administeredseparately simultaneously, before or after administration of nucleicacid molecules. In addition, other agents which may functiontransfecting agents and/or replicating agents and/or inflammatory agentsand which may be co-administered with a GVF include growth factors,cytokines and lymphokines such as a-interferon, gamma-interferon,GM-CSF, platelet derived growth factor (PDGF), TNF, epidermal growthfactor (EGF), ILA, IL-2, IL4, IL-6, IL-10, IL-12 and IL-15 as well asfibroblast growth factor, surface active agents such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl Lipid A (WL), muramyl peptides, quinoneanalogs and vesicles such as squalene and squalene, and hyaluronic acidmay also be used administered in conjunction with the genetic constructIn some embodiments, an immunomodulating protein may be used as a GVF.In some embodiments, the nucleic acid molecule is provided inassociation with PLG to enhance delivery/uptake.

The pharmaceutical compositions according to the present inventioncomprise about 1 nanogram to about 2000 micrograms of DNA. In somepreferred embodiments, pharmaceutical compositions according to thepresent invention comprise about 5 nanogram to about 1000 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 100 to about200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

According to some embodiments of the invention, methods of inducingimmune responses against an immunogen are provided by deliveringcompositions of the invention to an individual. The vaccine may be alive attenuated vaccine, a cell vaccine, a recombinant vaccine or anucleic acid or DNA vaccine.

In addition to using expressible forms of immunomodulating proteincoding sequence to improve genetic vaccines, the present inventionrelates to improved attenuated live vaccines and improved vaccines thatuse recombinant vectors to deliver foreign genes that encode antigens.Examples of attenuated live vaccines and those using recombinant vectorsto deliver foreign antigens are described in U.S. Pat. Nos. 4,722,848;5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 105,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668;5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,3 '64; 5,462,734;5,470,734; and 5,482,713, which are each incorporated herein byreference. Gene constructs are provided which include the nucleotidesequence that encodes an immunomodulating protein is operably linked toregulatory sequences that can function in the vaccine to effectexpression. The gene constructs are incorporated in the attenuated livevaccines and recombinant vaccines to produce improved vaccines accordingto the invention.

The present invention provides an improved method of immunizingindividuals that comprises the step of delivering gene constructs to thecells of individuals as part of vaccine compositions which include areprovided which include DNA vaccines, attenuated live vaccines andrecombinant vaccines. The gene constructs comprise a nucleotide sequencethat encodes an immunomodulating protein and that is operably linked toregulatory sequences that can function in the vaccine to effectexpression. The improved vaccines result in an enhanced cellular immuneresponse.

The present invention is useful to elicit enhanced immune responsesagainst a target protein, i.e. proteins specifically associated withpathogens, allergens or the individual's own “abnormal” cells. Thepresent invention is useful to immunize individuals against pathogenicagents and organisms such that an immune response against a pathogenprotein provides protective immunity against the pathogen. The presentinvention is useful to combat hyperproliferative diseases and disorderssuch as cancer by eliciting an immune response against a target proteinthat is specifically associated with the hyperproliferative cells. Thepresent invention is useful to combat autoimmune diseases and disordersby eliciting an immune response against a target protein that isspecifically associated with cells involved in the autoimmune condition.

According to some aspects of the present invention, DNA or RNA thatencodes a target protein and immunomodulating proteins is introducedinto the cells of tissue of an individual where it is expressed, thusproducing the encoded proteins. The DNA or RNA sequences encoding thetarget protein and one or both immunomodulating proteins are linked toregulatory elements necessary for expression in the cells of theindividual. Regulatory elements for DNA expression include a promoterand a polyadenylation signal. In addition, other elements, such as aKozak region, may also be included in the genetic construct.

In some embodiments, expressible forms of sequences that encode thetarget protein and expressible forms of sequences that encode bothimmunomodulating proteins are found on the same nucleic acid moleculethat is delivered to the individual.

In some embodiments, expressible forms of sequences that encode thetarget protein occur on a separate nucleic acid molecule from thenucleic acid molecules that contain expressible forms of sequences thatencode one or more immunomodulatory proteins. In. some embodiments,expressible forms of sequences that encode the target protein andexpressible forms of sequences that encode one or more of theimmunomodulatory proteins occur on a one nucleic acid molecule that isseparate from the nucleic acid molecule that contain expressible formsof sequences that encode one or more of the immunomodulating proteins.Multiple different nucleic acid molecules can be produced and deliveredaccording to the present invention and delivered to the individual. Forexample, in some embodiments, expressible forms of sequences that encodethe target protein occur on separate nucleic acid molecule from thenucleic acid molecules that contain expressible forms of sequences thatencode one or more of the two immunomodulating proteins which occur onseparate nucleic acid molecule from the nucleic acid molecules thatcontain expressible forms of sequences that encode one or moreimmunomodulating proteins. In such cases, all three molecules aredelivered to the individual.

The nucleic acid molecule(s) may be provided as plasmid DNA, the nucleicacid molecules of recombinant vectors or as part of the genetic materialprovided in an attenuated vaccine or cell vaccine. Alternatively, insome embodiments, the target protein and/or wither or bothimmunomodulating proteins maybe delivered as a protein in addition tothe nucleic acid molecules that encode them or instead of the nucleicacid molecules which encode them.

Genetic constructs may comprise a nucleotide sequence that encodes atarget protein or an immunomodulating protein operably linked toregulatory elements needed for gene expression. According to theinvention, combinations of gone constructs that include one thatcomprises an expressible form of the nucleotide sequence that encodes atarget protein and one that includes an expressible form of thenucleotide sequence that encodes an immunomodulating protein areprovided. Incorporation into a living cell of the DNA or RNA molecule(s)that include the combination of gene constructs results in theexpression of the DNA or RNA and production of the target protein andone or more immunomodulating proteins. An enhanced immune responseagainst the target protein results.

The present invention may be used to immunize an individual against allpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated such as viruses, and prokaryote such as gonorrhea, listeriaand shigella. In addition, the present invention is also useful toimmunize an individual against protozoan pathogens that include a stagein the life cycle where they are intracellular pathogens. Table 1provides a listing of some of the viral families and genera for whichvaccines according to the present invention can be made. DNA constructsthat comprise DNA sequences that encode the peptides that comprise atleast an epitope identical or substantially similar to an epitopedisplayed on a pathogen antigen such as those antigens listed on thetables are useful in vaccines. Moreover, the present invention is alsouseful to immunize an individual against other pathogens includingprokaryotic and eukaryotic protozoan pathogens as well as multicellularparasites such as those listed on Table 2.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material that encodes immunogenic proteins againstwhich a protective immune response can be mounted must be included in agenetic construct as the coding sequence for the target. Whether thepathogen infects intracellularly, for which the present invention isparticularly useful, or extracellularly, it is unlikely that allpathogen antigens will elicit a protective response. Because DNA and RNAare both relatively small and can be produced relatively easily, thepresent invention provides the additional advantage of allowing forvaccination with multiple pathogen antigens. The genetic construct usedin the genetic vaccine can include genetic material that encodes manypathogen antigens. For example, several viral genes may be included in asingle construct thereby providing multiple targets.

Tables 1 and 2 include lists of some of the pathogenic agents andorganisms for which genetic vaccines can be prepared to protect anindividual from infection by them. In some preferred embodiments, themethods of immunizing an individual against a pathogen are directedagainst HIV, HSV, HCV, WNV or HBV.

Another aspect of the present invention provides a method of conferringa protective immune response against hyperproliferating cells that arecharacteristic in hyperproliferative diseases and to a method oftreating individuals suffering from hyperproliferative diseases.Examples of hyperproliferative diseases include all forms of cancer andpsoriasis.

It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes—an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. To immunize against hyperproliferative diseases,a genetic construct that includes a nucleotide sequence that encodes aprotein that is associated with a hyperproliferative disease isadministered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides; which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein that is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used target antigens for autoimmune disease. Othertumor-associated proteins can be used as target proteins such asproteins that are found at higher levels in tumor cells including theprotein recognized by monoclonal antibody 17-IA and folate bindingproteins or PSA.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer inindividual. Using genetic screening and/or family health histories, itis possible to predict the probability a particular individual has fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of genetic constructs serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (1DDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs) thatare involved in the disease have been characterized. These TCRs includeVβ-3, Vβ-14, 20 Vβ-17 and Vα-17. Thus, vaccination with a DNA constructthat encodes at least one of these proteins will elicit an immuneresponse that will target T cells involved in RA. See: Howell, M. D., etal., 1991 Proc. Nat. Acad. Sci. USA 88:10921-10925; Piliard, X., et al,1991 Science 253:325-329; Williams, W. V., et al., 1992 J. Clin. Invest.90:326-333; each of which is incorporated herein by reference. In MS,several specific variable regions of TCRs that are involved in thedisease have been characterized. These TCRs include VfP and Va-10. Thus,vaccination with a DNA construct that encodes at least one of theseproteins will elicit an immune response that will target T cellsinvolved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et. al, 1990 Nature 345:344-346; eachof which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs that areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a DNA construct that encodes at least oneof these proteins will elicit an immune response that will target Tcells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes DNA constructsthat encode the variable region of such anti-DNA antibodies found in thesera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al, 1990 Proc. Natl. Acad Sci. USA87:1066, which is incorporated herein by reference.

The contents of the ASCII text file named SequenceLstg.txt (5 KB insize), which was submitted to the United States Patent and TrademarkOffice on Jun. 22, 2011, in accordance with 37 CFR §§1.821-1.825, areincorporated by reference into the specification in their entirety.

TABLE 1 Picornavirus Family Rhinoviruses: (Medical) responsible for -50% cases of the common cold. Genera: Etheroviruses: (Medical) includespolioviruses, coxsackieviruses, echoviruses, and human enterovirusessuch as hepatitis A virus. Apthoviruses: (Veterinary) these are the footand mouth disease viruses. Target antigens: VP1, VP2, VP3, VP4, VPGCalcivirus Family Norwalk Group of Viruses: (Medical) these viruses areGenera: an important causative agent of epidemic gastroenteritis.Togavirus Family Alphaviruses: (Medical and Veterinary) examples includeGenera: Senilis viruses, RossRiver virus and Eastern & Western Equineencephalitis. Reovirug: (Medical) Rubella virus. Flariviridue FamilyExamples include: (Medical) dengue, yellow fever, Japanese encephalitis,St. Louis encephalitis and tick borne encephalitis viruses. West Nilevirus (Genbank NC001563, AF533540, AF404757, AF404756, AF404755,AF404754, AF404753, AF481864, M12294, AF317203, AF196835, AF260969,AF260968, AF260967, AF206518 and AF202541) Representative Targetantigens: E NS5 C Hepatitis C Virus: (Medical) these viruses are notplaced in a family yet but are believed to be either a togavirus or aflavivirus. Most similarity is with togavirus family. CoronavirusFamily: Infectious bronchitis virus (poultry) (Medical and Veterinary)Porcine transmissible gastroenteric virus (pig) Porcine hemaglutinatingencephalomyelitis virus (pig) Feline infectious peritonitis virus (cats)Feline enteric coronavirus (cat) Canine coronavirus (dog) SARSassociated coronavirus The human respiratory coronaviruses cause ~40cases of common cold. EX. 224E, OC43 Note - coronaviruses may causenon-A, B or C hepatitis Target antigens: E1 - also called M or matrixprotein E2 - also called S or Spike protein E3 - also called BE orhemagglutin-elterose glycoprotein (not present in all coronaviruses) N -nucleocapsid Rhabdovirus Family Vesiliovirus Genera: Lyssavirus:(medical and veterinary) rabies Target antigen: G protein N proteinFiloviridue Family: Hemorrhagic fever viruses such as Marburg and Ebola(Medical) virus Paramyxovirus Family: Paramyxovirus: (Medical andVeterinary) Genera: Mumps virus, New Castle disease virus (importantpathogen in chickens) Morbillivirus: (Medical and Veterinary) Measles,canine distemper Pneuminvirus: (Medical and Veterinary) Respiratorysyncytial virus Orthomyxovirus Family (Medical) The Influenza virusBungavirus Family Bungavirus: (Medical) California encephalitis, LAGenera: Crosse Phlebovirus: (Medical) Rift Valley Fever Hantavirus:Puremala is a hemahagin fever virus Nairvirus (Veterinary) Nairobi sheepdisease Also many unassigned bungaviruses Arenavirus Family (Medical)LCM, Lassi fever virus Reovirus Family Reovirus: a possible humanpathogen Genera: Rotavirus: acute gastroenteritis in childrenOrbiviruses: (Medical and Veterinary) Colorado Tick fever, Lebombo(humans) equine encephalosis, blue tongue Retroyirus Family Sub-Family:Oncorivirinal: (Veterinary) (Medical) feline leukemia virus, HTLVI andHTLVII Lentivirinal: (Medical and Veterinary) HIV, felineimmunodeficiency virus, equine infections, anemia virus SpumavirinalPapovavirus Family Sub-Family: Polyomaviruses: (Medical) BKU and JCUviruses Sub-Family: Papillomavirus: (Medical) many viral typesassociated with cancers or malignant progression of papilloma.Adenovirus (Medical) EX AD7, ARD., O.B. - cause respiratory disease -some adenoviruses such as 275 cause enteritis Parvovirus Family(Veterinary) Feline parvovirus: causes feline enteritis Felinepanleucopeniavirus Canine parvovirus Porcine parvovirus HerpesvirusFamily Sub-Family: alphaherpesviridue Genera: Simplexvirus (Medical)HSVI (Genbank X14112, NC001806), HSVII (NC001798) Varicellovinis:(Medical Veterinary) pseudorabies - varicella zoster Sub-Family -betaherpesviridue Genera: Cytomegalovirus (Medical) HCMV MuromegalovirusSub-Family. Gammaherpesviridue Genera: Lymphocryptovirus (Medical) EBV -(Burkitts lympho) Rhadinovirus Poxvirus Family Sub-Family:Chordopoxviridue (Medical - Veterinary) Genera: Variola. (Smallpox)Vaccinia (Cowpox) Parapoxivirus - Veterinary Auipoxvirus - VeterinaryCapripoxvirus Leporipoxvirus Suipoxviru's Sub-Family: EntemopoxviridueHepadnavirus Family Hepatitis B virus Unclassified Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneurnococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella, melioidosis;, sahnonella; shigellosis;hemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella);streptobacillus mortiliformis and spirillum; listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria, cholera, anthrax; donovanosis(granuloma inguinale); and bartonellosis. Pathogenic anaerobic bacteriainclude: tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis; -treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis;. nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlarnydial infections include: mycoplasmapneurnoniae; lymphogranuloma venereum; psittacosis; and perinatalchlarnydial infections. Pathogenic eukaryotes Pathogenic protozoans andhelminths and infections thereby include: amebiasis; malaria;leishmaniasis; trypanosomiasis; toxoplasmosis; pneurnocystis carinii;babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis;nematodes; trematodes or flukes; and cestode (tapeworm) infections.

1. A nucleic acid molecule comprising SEQ ID NO:
 1. 2. The nucleic acidmolecule of claim 1 free of coding sequence for an IL-15 signalsequence.
 3. The nucleic acid molecule of claim 1 free of IL-15 Kozakregion and/or IL-15 5′ untranslated region and/or IL-15 3′ untranslatedregion.
 4. The nucleic acid molecule of claim 1 comprising a codingsequence for a non-IL-15 signal sequence.
 5. The nucleic acid moleculeof claim 1 further comprising coding sequence for an immunogen.
 6. Thenucleic acid molecule of claim 5 wherein said immunogen is a pathogenantigen, or a cancer-associated antigen.
 7. The isolated nucleic acidmolecule of claim 6 wherein said immunogen is a pathogen antigen from apathogen selected from the group consisting of HIV, HSV, HCV, and WNV.8. The isolated nucleic acid molecule of claim 1 wherein said isolatednucleic acid molecule is a plasmid.
 9. The nucleic acid molecule ofclaim 1 incorporated into a viral vector.
 10. The nucleic acid moleculeof claim 1 incorporated into a live attenuated pathogen.
 11. Acomposition comprising a nucleic acid molecule of claim 1 and a nucleicacid molecule that comprises a nucleic acid sequence that encodes animmunogen.
 12. The composition of claim 11 wherein said immunogen is apathogen antigen, or a cancer-associated antigen.
 13. The composition ofclaim 12 wherein said immunogen is a pathogen antigen from a pathogenselected from the group consisting of HIV, HSV, HCV, and WNV.
 14. Thecomposition of claim 11 wherein said nucleic acid molecules areplasmids.
 15. An injectable pharmaceutical composition comprising thenucleic acid molecules as in any one of claims 1-3, 5-9 or thecomposition of claim
 10. 16. A method of modulating an immune responsein an individual comprising administering to said individual a nucleicacid molecule of claim 1, wherein the nucleic acid molecule is operablylinked to a promoter and a polyadenylation signal.
 17. A recombinantvaccine comprising a nucleic acid sequence that encodes an immunogen anda nucleic acid sequence of claim
 1. 18. The recombinant vaccine of claim17 wherein said immunogen is a pathogen antigen, or a cancer-associatedantigen.
 19. The recombinant vaccine of claim 18 wherein said immunogenis a pathogen antigen from a pathogen selected from the group consistingof HIV, HSV, HCV, and WNV.
 20. The recombinant vaccine of claim 17wherein said recombinant vaccine is a recombinant vaccinia vaccine. 21.A method of inducing an immune response in an individual against animmunogen comprising administering to said individual a nucleic acidmolecule of claim 5, wherein the nucleic acid molecule is operablylinked to a promoter and a polyadenylation signal.
 22. A method ofinducing an immune response in an individual against an immunogencomprising administering to said individual a composition of claim 11,wherein the nucleic acid molecules of claim 12 are operably linked to apromoter and a polyadenylation signal.
 23. A method of inducing animmune response in an individual against an immunogen comprisingadministering to said individual a recombinant vaccine of claim 17,wherein the nucleic acids of claim 18 are operably linked to a promoterand a polyadenylation signal.
 24. The nucleic acid molecule of claim 1free of coding sequence for an IL-15 signal sequence and/or free ofIL-15 Kozak region and IL-15 5′ untranslated region and IL-153′untranslated region and comprising a coding sequence for a non-IL-15signal sequence.
 25. The nucleic acid molecule of claim 24 comprising acoding sequence for a non-IL-15 signal sequence operably linked to SEQID NO:1.
 26. The nucleic acid molecule of claim 1 wherein the nucleicacid molecule is a plasmid.
 27. The nucleic acid molecule of claim 5free of coding sequence for an IL-15 signal sequence and/or free ofIL-15 Kozak region and/or IL-15 5′ untranslated region and/or IL-153′untranslated region.
 28. The nucleic acid molecule of claim 27comprising a coding sequence for a non-IL-15 signal sequence operablylinked to SEQ ID NO:1.
 29. The nucleic acid molecule of claim 4 whereinthe nucleic acid molecule is a plasmid.
 30. The composition of claim 11wherein the nucleic acid molecule comprising SEQ ID NO: 1 is free ofcoding sequence for an IL-15 signal sequence and/or free of IL-15 Kozakregion and/or IL-15 5′ untranslated region and/or IL-15 3′ untranslatedregion.
 31. The composition of claim 30 wherein the nucleic acidmolecule comprising SEQ ID NO: 1 comprises a coding sequence for anon-IL-15 signal sequence operably linked to SEQ ID NO:1.